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. 2007 Oct 23;104(43):17081-6.
doi: 10.1073/pnas.0704126104. Epub 2007 Oct 16.

The EGF receptor is required for efficient liver regeneration

Anuradha Natarajan  1 , Bettina Wagner, Maria Sibilia
Affiliations

The EGF receptor is required for efficient liver regeneration

Anuradha Natarajan et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19656

Abstract

Mice lacking the EGF receptor (EGFR) die between midgestation and postnatal day 20 with various defects in neural and epithelial organs. Here, we generated mice carrying a floxed EGFR allele to inactivate the EGFR in fetal and adult liver. Perinatal deletion of EGFR in hepatocytes resulted in decreased body weight, whereas deletion in the adult liver did not affect body mass. Although liver function was not affected, after partial hepatectomy mice lacking EGFR in the liver showed increased mortality accompanied by increased levels of serum transaminases indicating liver damage. Liver regeneration was delayed in the mutants because of reduced hepatocyte proliferation. Analysis of cell cycle progression in EGFR-deficient livers indicated a defective G(1)-S phase entry with delayed transcriptional activation and reduced protein expression of cyclin D1 followed by reduced cdk2 and cdk1 expression. Impaired liver regeneration was accompanied by compensatory up-regulation of TNFalpha in the serum and prolonged activation of c-Jun. Moreover, p38alpha and NF-kappaB activation was reduced in regenerating mutant livers, indicating an impaired stress response after hepatectomy. Our studies demonstrate that EGFR is a critical regulator of hepatocyte proliferation in the initial phases of liver regeneration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of EGFRf/f mice. (A) The first line depicts the mouse genomic EGFR locus with the restriction sites and DNA probe used for Southern blot analysis. The targeting construct for homologous recombination in ES cells harbors a neomycin (neo) resistance cassette flanked by frt sites (black circles) for positive and a diphtheria toxin (DTα) cassette for negative selection. Cre-mediated recombination at the loxP sites (black triangles) generates the deleted EGFRΔ allele. C, XmaCI; E, EcoRI; H, HindIII; K, KpnI; S, SalI; X, XbaI. (B) Southern blot analysis of genomic DNA of electroporated ES clones showing the presence of the wild-type (+) and the EGFRf allele. (C) Southern blot analysis of offspring from crosses with Mox2-cre transgenic mice.
Fig. 2.
Fig. 2.
Analysis of mice lacking EGFR in the liver. (A) Body weight of EGFRf/f and EGFRΔhep mice at various times after birth. Results represent the mean ± SEM of six litters from five independent breeding cages. *, P < 0.05; **, P < 0.005. (B) Southern blot analysis of liver DNA of Alfp-cre mice at the indicated postnatal days. (C) Western blot analysis of EGFRf/f and EGFRΔhep livers at the indicated times after birth. Tubulin was used as loading control. (D) Southern blot analysis of livers of EGFRΔliv mice 3 days after the last pIpC injection. (E) Kaplan–Meier plot showing significant reduction in survival of EGFRΔliv (P = 0.033) and EGFRΔhep (P = 0.019) mice after PH. (F) Liver/body weight ratio in EGFRΔliv and EGFRΔhep mice 7 days after PH or sham surgery. Data indicate mean ± SEM of five mice per group. *, P < 0.05; **, P < 0.005.
Fig. 3.
Fig. 3.
Impaired proliferation after PH in mice lacking EGFR in the liver. (A–C) Proliferation measured by BrdU incorporation in EGFRf/f (A), EGFRΔliv (B), and EGFRΔhep (C) livers at 48 h after PH. Arrows point to BrdU-positive cells. (D) Quantification of BrdU-positive cells at the indicated times after PH. The results represent the mean ± SEM of six to seven independent livers. *, P < 0.05; **, P < 0.005.
Fig. 4.
Fig. 4.
Liver enzyme and cytokine production after PH. (A and B) GOT (A) and GPT (B) levels measured in the serum of control and EGFRΔliv mice at the indicated times after PH. (C and D) TNFα levels measured by ELISA in liver extracts (C) and serum (D) of EGFRΔliv and EGFRf/f mice at the indicated times after PH. The results represent mean ± SEM of five to six mice. *, P < 0.05. (D) Western blot analysis of membrane-bound TNFα in regenerating EGFRf/f and EGFRΔliv livers after PH with tubulin as loading control.
Fig. 5.
Fig. 5.
Cell cycle analysis of regenerating livers lacking the EGFR. (A) Cyclin D expression analyzed by RNase protection assay with L32 and GAPDH as loading controls in regenerating EGFRf/f and EGFRΔliv livers. (B) Expression of various cell cycle proteins in regenerating EGFRf/f and EGFRΔliv livers with tubulin as loading control. (C) Immunoprecipitated cyclin D1-associated kinase activity in EGFRf/f and EGFRΔliv livers after PH. (D and E) Quantification of cyclin D1 mRNA (D) and protein (E) expression at the indicated times after PH. The results represent the mean ± SEM of five independent livers. *, P < 0.05; **, P < 0.005.
Fig. 6.
Fig. 6.
Activation of signaling pathways in regenerating livers. Activation of c-Jun, p38, and NF-κB was analyzed by Western blot in regenerating EGFRf/f and EGFRΔliv livers with tubulin as loading control. Numbers below the p-Jun and Jun blot indicate the fold induction relative to the 0 time point after normalizing the values with total Jun and tubulin, respectively.
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